New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil

It is generally accepted that there are two major centers of genetic diversification of common beans (Phaseolus vulgaris L.): the Mesoamerican (Mexico, Colombia, Ecuador and north of Peru, probably the primary center), and the Andean (southern Peru to north of Argentina) centers. Wild common bean is not found in Brazil, but it has been grown in the country throughout recorded history. Common bean establishes symbiotic associations with a wide range of rhizobial strains and Rhizobium etli is the dominant microsymbiont at both centers of genetic diversification. In contrast, R. tropici, originally recovered from common bean in Colombia, has been found to be the dominant species nodulating field-grown common-bean plants in Brazil. However, a recent study using soil dilutions as inocula has shown surprisingly high counts of R. etli in two Brazilian ecosystems. In the present study, RFLP-PCR analyses of nodABC and nifH genes of 43 of those Brazilian R. etli strains revealed unexpected homogeneity in their banding patterns. The Brazilian R. etli strains were closely similar in 16S rRNA sequences and in nodABC and nifH RFLP-PCR profiles to the Mexican strain CFN 42^T, and were quite distinct from R. etli and R. leguminosarum strains of European origin, supporting the hypothesis that Brazilian common bean and their rhizobia are of Mesoamerican origin, and could have arrived in Brazil in pre-colonial times. R. tropici may have been introduced to Brazilian soils later, or it may be a symbiont of other indigenous legume species and, due to its tolerance to acidic soils and high temperature conditions became the predominant microsymbiont of common bean.     

Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.     

Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: Selection of native strains that replace conventional N fertilization

Phaseolus vulgaris is a legume extensively cultivated in Spain, León province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Riñón” in several soils from León province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.     

Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures

Strains of Rhizobium tropici IIB, CIAT899 and F98.5, both showing good N₂ fixation, and a R. etli strain W16.3SB were introduced into a field which had no history of bean culture. Plant dilution estimates showed that in the presence of its host (Phaseolus vulgaris cv. Carioca) during the cropping seasons and the subsequent fallow summer periods, the bean rhizobial populations increased from less than 30 to 10³ g^–1 dry soil after 1 year and to 10⁴ g^–1 dry soil after 2 years. In the 1st year crop, the inoculated strains occupied most of the nodules, which resulted in a higher nodulation and C₂H₂ reduction activity. Without reinoculation for the second and third crops, however, little R. tropici IIB was recovered from the nodules and the bean population consisted mainly of R. etli, R. leguminosarum bv. phaseoli, and R. tropici IIA. Reinoculation with our superior R. tropici IIB strains before the second crop resulted in R. tropici IIB occupying the main part of the nodules and a positive effect on nodulation and C₂H₂ reduction activity, but reintroduction of the inoculant strain in the third season did not have any effect.     

Polyphasic characteristics of bradyrhizobia isolated from nodules of peanut (Arachis hypogaea) in China

Peanuts (Arachis hypogaea L.) were introduced to China about 500 years ago. However, the diversity of Rhizobial strains in China that can nodulate peanut was poorly understand. Diversity and phylogeny of 50 slow-growing strains, isolated from root nodules of peanut in different geographical regions of China, were studied using polyphasic techniques. All stains were clustered by phenotypic tests into two distinct groups: Group I: 16S rRNA RFLP genotype 3, and Group II, which divided into 16S rRNA RFLP genotypes 1 and 2. Genotype 1 shares the same genotype with USDA110, USDA122 and USDA127 of Bradyrhizobium japonicum, and genotype 2 solely consisted of extra-slow growing bradyrhizobia isolated from Hongan, China. Results of 16S rRNA sequencing revealed that peanut bradyrhizobia were phylogenetically related to B. japonicum and their sequence divergence was less than 1.1%. Based upon the size of the internally transcribed spacer (ITS) between the16S and 23S RNA genes, strains were classified into ITS-I, ITS-II and ITS-III genotypes. Strains could be further divided into sub-clusters IA, IB, IIa, IIb and IIc five sub-clusters through ITS PCR-RFLP and repetitive extragenic palindromic PCR (REP-PCR) analysis. Host specificity test revealed that all peanut bradyrhizobia tested nodulated Phaseolus vulgaris and strains of clusters IIb and IIc nodulated Glycine soja efficiently. Bradyrhizobia isolated from peanut were related, but still exhibited phylogenetical divergence with B. japonicum.     

Nodulation and growth of common bean (Phaseolus vulgaris) under water deficiency

Three experiments were conducted in order to investigate the effect of water deficiency on nodulation, rhizobial diversity and growth of common bean. In the first experiment, the effect of water deficiency was studied on two soil samples under glasshouse conditions. A significant decrease in nodulation and shoot dry weight production was observed. The molecular characterization of the root nodule isolates by PCR-RFLP of 16S rRNA and nodC genes showed that the nodulation by Rhizobium etli was severely inhibited. The in vitro analysis of salt tolerance indicated that drought stress favoured nodulation by salt-tolerant strains. In the second experiment, the effect of water deficiency was studied on sterilized sand using Rhizobium tropici CIAT899^T and Ensifer meliloti bv. mediterranense 4H41 as inoculants. The results showed that strain 4H41, which is the more salt tolerant, was more competitive and more effective under water deficiency than strain CIAT899^T. In the third experiment, the strain 4H41 was used to inoculate four fields. A significant increase in nodule number, shoot dry weight and grain yield was observed even in the non-irrigated soils. This work constitutes the first report of a strain enhancing the growth and the grain yield of common bean under water deficiency.     

Nodulation competitiveness of Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici strains measured by glucuronidase (gus) gene fusion

Summary The nodulation competitiveness of 17 Rhizobium leguminosarum bv. phaseoli and 3 R. tropici strains was analysed in growth pouches, at pH 5.2 and 6.4. All 20 strains were coinoculated with a gus ⁺ strain of R. leguminosarum bv. phaseoli strain KIM5s. The gus⁺ phenotype, carrying the glucuronidase gene, was used to type nodules directly in the growth pouches. Nodule occupancy ranged from 4% for the least competitive to 96% for the most competitive R. leguminosarum bv. phaseoli strain. The R. tropici strains showed low rates of nodule occupancy at pH 6.4 but their competitiveness improved significantly under acid conditions. CIAT 895 was the only R. leguminosarum bv. phaseoli strain that was less competitive (P<0.05) at the lower pH. The competitiveness of all the other R. leguminosarum bv. phaseoli strains was unaffected by pH. Various physiological and genetic properties of the strains were analysed in search of correlations with nodulation competitiveness. Hybridisation patterns with three different DNA probes (nif KDH, common nod genes, and hup genes) and the metabolism of 53 different C sources were compared. No general correlations were found between hybridisation or growth pattern and competitiveness. The less competitive R. tropici strains had a unique DNA hybridisation pattern and were not able to use shikimate, ferulate, coumarate, or asparagine as C sources. Most of the less competitive R. leguminosarum bv. phaseoli strains could not metabolize either ferulate or coumarate. This might indicate a relationship between nodulation competitiveness and the ability to degrade aromatic compounds.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

The abundance and efficacy of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

For optimum production, the use of commercial rhizobial inoculant on pea (Pisum sativum L.) at seeding is necessary in the absence of compatible rhizobial strains or when rhizobial soil populations are low or symbiotically ineffective. Multiple site experiments were conducted to characterize the abundance and effectiveness of resident populations of Rhizobium leguminosarum bv. viciae (Rlv) in eastern Canadian prairie soils. A survey of 20 sites across a broad geographical range of southern Manitoba was carried out in 1998 and was followed by more intensive study of five of the sites in 1999 and 2000. Appreciable nodulation of uninoculated pea was observed at all sites which had previously grown inoculated pea. However, uninoculated pea grown at two sites, which had not previously grown pea, had negligible nodulation. Likewise, wild Lathyrus sp. and Vicia sp. plants collected from uncultivated areas adjacent to agricultural sites were poorly nodulated. In the more intensively studied sites, there was a tendency towards higher nodulation in pea plants receiving commercial inoculant containing Rlv strain PBC108 across all site-years (e.g., 4.7% in nodulation and 22% in nodule mass), but the effect was significant at only 2 of 10 site-years. Despite a relatively high range of soil pH (6-8), regression analysis indicated that decreasing soil pH resulted in lower nodulation rates. Likewise, electrical conductivity (EC) was correlated to nodulation levels, however the effect of EC was likely more indicative of the influence of soil texture and organic matter than salinity. As with nodulation, commercial inoculation tended to increase above-ground dry matter (DM) and fixed-N (estimated by the difference method) at the early pod-filling stage, but again the effects were significant at only 2 of 10 site-years. Specifically, above-ground DM and fixed-N levels were up to 29 and 51% greater, respectively, in inoculated compared to non-inoculated treatments at these sites. Addition of N-fertilizer at a rate of 100 kg N ha⁻¹ decreased nodulation at almost all site-years (by as much as 70% at one site), but rarely resulted in increases in above-ground DM compared to inoculated plots. The study indicates for the first time that populations of infective, and generally effective strains of Rlv occur broadly in agricultural soils across the eastern Canadian prairie, but that there is a tendency for increased symbiotic efficiency with the use of commercial inoculant.     

Rhizobia phylogenetically related to common bean symbionts Rhizobium giardinii and Rhizobium tropici isolated from peanut nodules in Central Argentina

Our previous studies of the native rhizobial population associated with peanut nodules in the Córdoba soils of Argentina revealed that this population is highly diverse and includes slow- and fast-growing isolates. The native fast-growing isolates NCHA22 and NET30 were selected on the basis of their plant growth promoting properties and their chromosomal genotypes were determined by 16S rDNA sequencing. NCHA22 and NET30 16S rDNA alleles were found to cluster with those of Rhizobium tropici group IIB and Rhizobium giardinii bv. giardinii strain H152, respectively. We have now characterized these isolates by analyzing the glnA and nifH genes to clarify their taxonomic position. These studies confirmed that fast-growing isolates belonging to species earlier described as bean symbionts were obtained from nodules of a leguminous plant that has been described as efficiently nodulated exclusively by slow-growing rhizobial strains.     

Rhizobia nodulating African Acacia spp. and Sesbania sesban trees in southern Ethiopian soils are metabolically and genomically diverse

The diversity of 110 rhizobial strains isolated from Acacia abyssinica, A. seyal, A. tortilis, Faidherbia albida, Sesbania sesban, Phaseolus vulgaris, and Vigna unguiculata grown in soils across diverse agro-ecological zones in southern Ethiopia was assessed using the Biolog™ system and amplified fragment length polymorphism (AFLP) fingerprinting technique. By cluster analysis of the metabolic and genomic fingerprints, the test strains were grouped into 13 Biolog and 11 AFLP clusters. Twenty-two strains in the Biolog method and 15 strains in the AFLP analysis were linked to eight and four reference species, respectively, out of the 28 included in the study. Most of the test strains (more than 80% of 110) were not related to any of the reference species by both methods. Forty-six test strains (42% of 110) were grouped into seven corresponding Biolog and AFLP clusters, suggesting that these groups represented the same strains, or in some cases clonal descendants of the same organisms. In contrast to the strains from S. sesban, isolates from Acacia spp. were represented in several Biolog and AFLP clusters indicating the promiscuous nature of the latter and widespread occurrence of compatible rhizobia in most of the soil sampling locations. The results showed that indigenous rhizobia nodulating native woody species in Ethiopian soils constituted metabolically and genomically diverse groups that are not linked to reference species.     

Novel strains of nodulating Burkholderia have a role in nitrogen fixation with papilionoid herbaceous legumes adapted to acid, infertile soils

We investigated the taxonomic position and symbiotic capabilities of two root-nodule bacterial strains isolated from the South African herbaceous, papilionoid legume Rhynchosia ferulifolia. The 16S rRNA gene sequence of the two strains was determined along with intragenic sequences of nodA and nifH, together with their symbiotic capabilities when inoculated onto the papilionoid legumes R. ferulifolia, Rhynchosia caribaea, Rhynchosia minima and Macroptilium atropurpureum (Siratro). Burkholderia phymatum STM815^T, Cupriavidus taiwanensis LMG 19424^T and root-nodule bacteria isolated from R. minima and Rhynchosia totta were included in the study. Root-nodule bacteria isolated from R. ferulifolia, WSM3937 and WSM3930, belong to the genus Burkholderia and are most closely related to Burkholderia terricola (98.8% similarity). The phylogenetic analysis of nodA and nifH revealed substantial similarity of the novel strains with Burkholderia tuberum STM678^T, a β-rhizobium also originated from South Africa, and only a distant relationship with South American Mimosa-nodulating β-rhizobia. R. ferulifolia was effectively nodulated only by Burkholderia sp. WSM3937 and WSM3930 and not by bradyrhizobia isolated from Rhynchosia minima and Rhynchosia totta or STM815 and LMG 19924. Nodules induced by the novel strains were determinate and hosted well organized symbiosomes within infected cells. In this study we describe a new symbiotic N-fixing relationship between Burkholderia sp. and the South African legume R. ferulifolia. This is the first report of N-fixation between β-rhizobia and an herbaceous, papilionoid legume from which the strains were originally isolated. The level of N-fixation in this symbiosis approached that achieved by effectively nodulated Medicago sativa and suggests that the β-rhizobia may have a role in N-fixation in agricultural systems.     

Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×10³ rhizobia g⁻¹ of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g⁻¹ soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N₂ fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5-9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N₂ fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N₂ fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.     

Plant growth promoting properties of a strain of Enterobacter ludwigii isolated from Lolium perenne rhizosphere

The capability of native bacterial strains isolated from Lolium perenne rhizosphere to behave as plant growth promoting bacteria and /or biocontrol agents was investigated. One strain (BNM 0357) over 13 isolates from the root tips of L. perenne resulted proved to be nitrogenase positive (ARA test) and an IAA producer. Conventional tests and the API 20E diagnostic kit indicated that BNM 0357 behaves to the Enterobacteriaceae family and to the Enterobacter genus. Molecular identification by 16S rRNA sequence analysis indicated that BNM 0357 had the highest similarity to Enterobacter ludwigii (EN-119). Isolate BNM 0357 had the capability to solubilize calcium triphosphate and to antagonize Fusarium solani mycelial growth and spore germination. Strain BNM 0357 also showed the ability to improve the development of the root system of L. perenne. This study disclosed features of E. ludwigii BNM 0357 that deserve further studies aimed at confirming its putative importance as a PGPR.     

Biological control of damping-off caused by Rhizoctonia solani using chitinase-producing Paenibacillus illinoisensis KJA-424

A bacterium having strong chitinolytic activity was isolated from a coastal soil in Korea and identified as Paenibacillus illinoisensis KJA-424 on the basis of the nucleotide sequence of a 16S rRNA gene. By activity staining after SDS-PAGE, three major chitinase bands with chitinolytic activity, approximate molecular weight of 63, 54 and 38 kDa were detected. On co-culture Rhizoctonia solani with KJA-424, abnormal swelling and deformation of R. solani hyphae were observed, where the release of N-acetyl-d-glucosamine was detected. The bacterium suppressed the symptom of damping-off cucumber seedlings caused by R. solani, in greenhouse trial.     

Evaluation of the intrinsic competitiveness and saprophytic competence of Rhizobium tropici IIB strains

Inoculation of beans (Phaseolus vulgaris L.) with strains of R. tropici IIB and R. etli resulted in the disappearance of the R. tropici IIB stains from the nodule population and their replacement by other (non R. tropici IIB) bean symbionts (Vlassak et al. 1996). Coinoculation studies in monoxenic conditions and in soil core microcosms with plants harvested at two different growth stages indicated that the inoculated R. tropici IIB strains CIAT899 and F98.5 possess a good intrinsic competitiveness which declines, however, at a later plant growth stage and in soil conditions. The poor saprophytic competence of R. tropici IIB strain CIAT899 was further demonstrated by its poor survival in soil core microcosms after bean harvest. Strains were isolated from the field plots with a 3-year bean-planting history, characterized and evaluated for their competitiveness against R. tropici IIB strain CIAT899. Isolates from field plots, which had been repeatedly inoculated with R. tropici IIB strain CIAT899, showed a higher nodule occupancy compared to R. tropici IIB strain CIAT899, and this higher competitiveness exhibited by the field isolates might be an additional reason for the poor performance of R. tropici IIB strain CIAT899 in the field study. Plots with and without a history of bean production revealed after 3-year bean cultivation an almost totally different population that also significantly differed in competitiveness. Received: 12 February 1996     

Genetic diversity among Elaeagnus compatible Frankia strains and sympatric-related nitrogen-fixing actinobacteria revealed by nifH sequence analysis

Elaeagnus compatible Frankia isolates from Tunisian soil have been previously clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic subgroups, while strain BMG5.6 was described as a new lineage closely related to Frankia and Micromonospora genera. In this study we further assess the diversity of captured Frankia and the relationship with BMG5.6-like actinobacteria, by using nifH gene sequences. Using PCR-RFLP screening on DNA extracted from lobe nodules, additional microsymbionts sharing BMG5.6 features have been detected proving a widespread occurrence of these actinobacteria in Elaeagnus root nodules. Neighbour-Joining trees of Frankia nifH sequences were consistent with previously published 16S rRNA and GlnII phylogenetic trees. Although four main clades could be discerned, actinobacterial strain BMG5.6 was clustered with Frankia strains isolated from Elaeagnus. The present study underscored the emanation of new diazotrophic taxon isolated from actinorhizal nodules occupying intermediate taxonomic position between Frankia and Micromonospora. Moreover, its aberrant position in nifH phylogeny should open network investigations on the natural history of nitrogen-fixing gene among actinobacteria.     

Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions

This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.     

Diversity and antagonistic potential of Pseudomonas spp. associated to the rhizosphere of maize grown in a subtropical organic farm

Pseudomonas spp. are one of the most important bacteria inhabiting the rhizosphere of diverse crop plants and have been frequently reported as biological control agents (BCAs). In this work, the diversity and antagonistic potential of Pseudomonas spp. in the rhizosphere of maize cultivars Nitroflint and Nitrodent grown at an organic farm in Brazil was studied by means of culture-dependent and -independent methods, respectively. Sampling of rhizosphere soil took place at three different stages of plant development: 20, 40 and 106 days after sowing. A PCR-DGGE strategy was used to generate specific Pseudomonas spp. fingerprints of 16S rRNA genes amplified from total community rhizosphere DNA. Shifts in the relative abundance of dominant populations (i.e. PCR-DGGE ribotypes) along plant development were detected. A few PCR-DGGE ribotypes were shown to display cultivar-dependent relative abundance. No significant differences in diversity measures of DGGE fingerprints were observed for different maize cultivars and sampling times. The characterisation and assessment of the antagonistic potential of a group of 142 fluorescent Pseudomonas isolated from the rhizosphere of both maize cultivars were carried out. Isolates were phenotypically and genotypically characterised and screened for in vitro antagonism towards three phytopathogenic fungi and the phytopathogenic bacterium Ralstonia solanacearum. Anti-fungal activity was displayed by 13 fluorescent isolates while 40 isolates were antagonistic towards R. solanacearum. High genotypic and phenotypic diversity was estimated for antagonistic fluorescent Pseudomonas spp. PCR-DGGE ribotypes displayed by antagonists matched dominant ribotypes of Pseudomonas DGGE fingerprints, suggesting that antagonists may belong to major Pseudomonas populations in the maize rhizosphere. Antagonists differing in their genotypic and phenotypic characteristics shared the same DGGE electrophoretic mobility, indicating that an enormous genotypic and functional diversity might be hidden behind one single DGGE band. Cloning and sequencing was performed for a DGGE double-band which had no corresponding PCR-DGGE ribotypes among the antagonists. Sequences derived from this band were affiliated to Pseudomonas stutzeri and P. alcaligenes 16S rRNA gene sequences. As used in this study, the combination of culture-dependent and -independent methods has proven to be a powerful tool to relate functional and structural diversity of Pseudomonas spp. in the rhizosphere.